Abnormalities in extrinsic and intrinsic innervation and in the abundance and distribution of interstitial cells of Cajal are known to contribute to impairment of gastrointestinal motility in diabetes. Abnormalities in smooth muscle function in relation to diabetes have not been fully explored. The proposed studies are intended to fill this gap in our knowledge. Our previous studies showed that sustained smooth muscle contraction and relaxation are regulated by the RhoA/Rho kinase and cGMP/PKG pathways, respectively. Our preliminary studies in diabetic animal models (NOD and db/db mice) and human diabetic smooth muscle have shown that hyperglycemia causes upregulation of the RhoA/Rho kinase pathway and downregulation of the cGMP/PKG pathway leading to sustained increase in smooth muscle contraction and decrease in its ability to relax. The focus of this proposal is to define the molecular mechanisms that trigger these changes in smooth muscle signaling and identify their contribution to motility dysfunction in diabetes. 1) Microarray and RNA hybrid analysis of control and diabetic gastric and colonic smooth muscle revealed a functional link between miR-133a and RhoA expression that led us to hypothesize that a decrease in miR-133a expression in diabetic smooth muscle mediates the increase in RhoA expression leading to upregulation of the RhoA/Rho kinase pathway and sustained increase in muscle contraction (Specific Aim 1). 2) Sequence analysis of the human and mouse PDE5 promoter region in gastric and colonic smooth muscle identified a link to transcription factor NFATc4 that led us to hypothesize that O- GlcNAcylated phospholamban mediates sequential activation of calcineurin and NFATc4 and increase in PDE5 expression leading to downregulation of the cGMP/PKG pathway and inhibition of muscle relaxation (Specific Aim 2). 3) Preliminary studies identified a link between miR-21 and a decrease in cystathionine- -lyase (CSE) expression in diabetic smooth muscle that led us to hypothesize that a decrease in endogenous H2S formation attenuates inhibitory S-sulfhydration of RhoA and PDE5 and promotes upregulation of the RhoA/Rho kinase and downregulation of the cGMP/PKG pathways (Specific Aim 3). The novel concepts underlying these hypotheses have been validated by preliminary studies in smooth muscle of human and diabetic NOD and db/db mice and support the notion that specific alterations in smooth muscle signaling are driven by high glucose levels. A novel approach involving intraperitoneal injection of miR-133a, miR-21, and other specific inhibitors so as to block selectively each pathway provided evidence that these specific alterations in smooth muscle signaling contribute to motility dysfunction in diabetes. Completion of these studies will expand our understanding of overall motility dysfunction in diabetes and offer avenues for the development of novel therapies.